Bacterial pathogens often damage their host through the action of toxins. Bacterial toxins modify host targets, primarily proteins, through covalent and non-covalent mechanisms and have several structure-function organizations, including exotoxins and type III secreted cytotoxins. While exotoxins often act at a distance from the site of infection, type III cytotoxins are delivered directly into the intoxicated host cell by the bacterium. The aims of this application have been to characterize the substrate recognition and intracellular trafficking of bacterial toxins. During the previous period, studies on the type III cytotoxins of Pseudomonas aeruginosa determined substrate recognition mechanisms of ExoS and ExoT and showed that intracellular trafficking within host cells increased the potency of ExoS for Ras and Rho GTPases. A translational project was also developed to detect initial PA infections in humans. This renewal will continue to characterize the intracellular trafficking and substrate recognition on ExoS and initiate structure-function studies on tetanus neurotoxin (TeNT). The proposed studies will characterize specific properties of ExoS and TeNT that define unique aspects of bacterial toxin action. Analyses utilize biochemical- and quantitative cell biological- approaches that allow corroborative analyses from the test tube to the cell. Studies on ExoS will characterize how intracellular trafficking within host cells increases the potency of ExoS and determine the mechanism that ADP-ribosylation inhibits signaling by small GTPases, like Ras and Rab5. Recent studies determined that gangliosides are the functional receptors of TeNT in neurons. This identification allows a fundamental question of neurotoxin pathology to be addressed, how does TeNT elicit spastic paralysis in the host? Studies on TeNT will characterize the intracellular trafficking of TeNT in neurons and determine how TeNT cleaves the SNARE protein, VAMP2. These studies define mechanistic properties of toxins that may lead to translational products for the detection, prevention, and intervention of bacterial infections.